丹参酮ⅡA对雷帕霉素诱导自噬M2巨噬细胞共培养的皮肤黑素瘤A375细胞EMT的影响  

The mechanism of TanshinoneⅡA on regulating EMT in cutaneous melanoma A375 cells through TAMs autophagy

作　　者：韩昭[1] 高艳玲 李志锋[1] 张建忠[1] 张伶[1] 刘钊 徐艳艳 李小静[1] HAN Zhao;GAO Yanling;LI Zhifeng;ZHANG Jianzhong;ZHANG Ling;LIU Zhao;XU Yanyan;LI Xiaojing(Department of Dermatology,Affiliated Hospital of Hebei Engineering University;Department of Emergency,Handan Central Hospital,Handan 056002,China)

机构地区：[1]河北工程大学附属医院皮肤科 [2]河北工程大学邯郸市中心医院急诊科,河北邯郸056002

出　　处：《皮肤性病诊疗学杂志》2022年第4期291-298,共8页Journal of Diagnosis and Therapy on Dermato-venereology

基　　金：河北省高等学校科学技术研究项目(ZD2020305)。

摘　　要：目的通过建立肿瘤相关巨噬细胞(TAMs)自噬水平改变对与之共培养的A375细胞的体系,进一步明确TAMs自噬在调控A375细胞上皮间质转化(EMT)及迁移侵袭过程中的作用。方法用PMA和IL-4相继作用于人单核细胞THP-172 h诱导其成M2型,流式细胞术及免疫荧光检测鉴定M2表面CD68、CD204、CD206等标志性分子表达情况,鉴定TAMs极化效率。用自噬调节剂雷帕霉素干预TAMs(M2)24 h,去除自噬调节药物的干预48 h后,通过蛋白免疫印迹方法以及免疫荧光检测各组细胞LC3-Ⅱ和Beclin-1表达,确定药物干预后的TAMs自噬水平。将雷帕霉素干预后的TAMs与人黑素瘤A375细胞进行非接触共培养,48 h后加入不同浓度TanⅡA 1 mg/L(低浓度)组、4 mg/L(高浓度)组,继续培养48 h后,Western blot检测A375细胞EMT相关因子的表达,并通过transwell检测A375细胞的侵袭和迁移能力。结果人单核细胞THP-1经PMA和IL-4先后作用共72 h后,细胞状态由半悬浮变为贴壁,经流式细胞术、免疫荧光检测发现M2表面CD68、CD204、CD206的表达较M0组明显增强,差异具有统计学意义(均P<0.05)。用自噬调节剂雷帕霉素干预TAMs 24 h后,免疫荧光、Western blot检测显示,LC3-Ⅱ及Beclin-1的表达明显上调,差异具有统计学意义(均P<0.05)。TAMs和A375细胞非接触共培养48 h后,不同浓度的TanⅡA作用于细胞,Western Blot检测显示,相对于空白组,TanⅡA低浓度组和高浓度组Beclin1、LC3-Ⅱ和E-cadherin蛋白表达均显著升高,N-cadherin蛋白表达均显著降低,组间差异均具有统计学意义(均P<0.05)。transwell实验显示,TanⅡA低浓度组和TanⅡA高浓度组处理后的A375细胞,其迁移、侵袭能力均有所下降,组间差异具有统计学意义(均P<0.05)。结论雷帕霉素可以诱导TAMs发生自噬,TanⅡA可以抑制自噬TAMs与之共培养的黑素瘤A375细胞的EMT过程,从而抑制其侵袭和转移。Objective To further clarify the role of TAMs autophagy in regulating the EMT and migration and invasion of A375 cells,the effects of TAMs autophagy changes on the biological behaviors of A375 cells co-cultured with TAMs were studied.Methods Human monocyte THP-1 cells were induced into M2 type by PMA and IL-4 for 72 h.The expressions of CD68,CD204 and CD206 on M2 surface were determined by flow cytometry and immunofluorescence,and the polarization efficiency of TAMs was determined.TAMs was treated with rapamycin,an autophagy modulator,for 24 h,and the expressions of LC3-Ⅱand Beclin-1 were detected by Western blot and immunofluorescence after the removal of autophagy modulator intervention for 48 h.The autophagy level of TAMs after drug intervention was determined.Non-contact co-culture was conducted between TAMs after rapamycin intervention and human melanoma A375 cells.Routine culture in the above incubator was conducted by adding low concentration TanⅡA group(1 mg/L)and high concentration TanⅡA group(4 mg/L).After 48 hours of culture,Western blot was used to detect the expression of EMT-related factors in A375 cells,and transwell was used to detect the invasion and migration of A375 cells.Results Human monocyte(THP-1)was treated with PMA and IL-4 for 72 h.Flow cytometry and immunofluorescence detection showed that the expressions of CD68,CD204 and CD206 on M2 surface were significantly increased compared with M0 group,and the difference was statistically significant(all P<0.05).After treated with rapamycin,the expressions of LC3-Ⅱand Beclin-1 were significantly up-regulated by immunofluorescence and Western blot analysis,with statistical significance(all P<0.05).After non-contact co-culture of TAMs and A375 cells for 48 h,the cells were treated with different concentrations of TanⅡA.Western Blot analysis showed that compared with the blank group,the protein expressions of Beclin1,LC3-Ⅱand E-cadherin in the low-concentration TanⅡA group and the high-concentration TanⅡA group were significantly in

关 键 词：TAMS 自噬 皮肤黑素瘤 丹参酮ⅡA EMT 

分 类 号：R965[医药卫生—药理学]